Production of a parvovirus vaccine in plants as viral coat protein fusions

ABSTRACT

The present invention relates to foreign peptide sequences fused to recombinant plant viral structural proteins and a method of their production. Fusion proteins are economically synthesized in plants at high levels by biologically contained tobamoviruses. The fusion proteins of the invention have are useful as antigens for inducing the production of antibodies having desired binding properties, e.g., protective antibodies, or for use as vaccine antigens for the induction of protective immunity against the parvovirus. Feline parvovirus epitopes were fused to the N-terminus of the TMV coat protein, expressed in Nicotiana plants, extracted, purified, characterized and administered to animals, resulting in protective immunity.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The present invention relates to the field of geneticallyengineered peptide production in plants, more specifically, to the useof tobamovirus vectors to express fusion proteins, and morespecifically, to a vaccine comprising an encapsidated virus having amodified coat protein which displays a parvovirus antigen.

[0003] 2. Description of the Background Art

[0004] Peptides are a diverse class of molecules having a variety ofimportant chemical and biological properties. Some examples include;hormones, cytokines, immunoregulators, enzyme inhibitors, vaccineantigens, adhesion molecules, receptor binding domains, and the like.The cost of chemical synthesis limits the potential applications ofsynthetic peptides for many uses such as therapeutic drugs or vaccines.There is a need for inexpensive and rapid synthesis of milligram andlarger quantities of naturally occurring polypeptides. Towards this goalmany animal and bacterial viruses have been used successfully as peptidecarriers.

[0005] The safe and inexpensive culture of plants provides anadvantageous alternative for cost-effective production ofpharmaceutically useful peptides. During the last decade, considerableprogress has been made in expressing foreign genes in plants. Foreignproteins are now routinely produced in many plant species either formodification of the plant or for protein extraction and production.Animal proteins have been effectively produced in plants (reviewed inKrebbers, E. et al., In: Plant Protein Engineering (P. R. Shewry et al.,eds.), Cambridge University Press, Cambridge, 1992, pp. 316-324).

[0006] Vectors for the genetic manipulation of plants have been derivedfrom several naturally occurring plant viruses, including tobacco mosaicvirus (TMV). TMV is the type member of the tobamovirus group. TMV hasstraight tubular virions of approximately 300×18 nm with a 4 nm-diameterhollow canal, consisting of approximately 2000 units of a single capsidprotein wound helically around a single RNA molecule. Virion particlesare 95% protein and 5% RNA by weight. The genome of TMV is composed of asingle-stranded RNA of 6395 nucleotides containing five large openreading frames (ORFs). Expression of each gene is regulatedindependently. The virion RNA serves as the messcenger RNA (mRNA) forthe 5′ genes, encoding the 126 kDa replicase subunit and the overlapping183 kDa replicase subunit that is produced by read-through of a UAG stopcodon approximately 5% of the time. Expression of the internal genes iscontrolled by different promoters on the minus-sense RNA that directsynthesis of 3′-coterminal subgenomic mRNAs which are produced duringreplication (FIG. 1). A detailed description of tobamovirus geneexpression and life cycle can be found, among other places, in Dawsonand Lehto, Adv. Vir. Res. 38:307-342 (1991). It is of interest toprovide new and improved vectors for the genetic manipulation of plants.

[0007] For production of specific proteins, transient expression offoreign genes in plants using virus-based vectors has severaladvantages. Products of plant viruses are among the highest producedproteins in plants. Often a viral gene product is the major proteinproduced in plant cells during virus replication. Many viruses are ableto spread quickly from an initial infection site to almost all cells ofthe plant. For these reasons, plant viruses have been developed intoefficient transient expression vectors for foreign genes in plants.Viruses of multi-cellular plants are relatively small, probably due tothe size limitation in the pathways that allow viruses to move toadjacent cells in systemic infection of the entire plant. Most plantviruses have single-stranded RNA genomes of less than 10 kb. Geneticallyaltered plant viruses provide one efficient means of transfecting plantswith genes encoding peptide-carrier fusion proteins. A discussion of TMVcoat protein fusions is provided in Turpen et al., U.S. Pat. No.5,977,438 entitled “Production of Peptides in Plants as Viral CoatProtein Fusions.” Nov. 2, 1999. See also: Yusibov V. et al., Proc. Natl.Acad. Sci. U.S.A 94:5784-5788 (1997); Modelska, A et al., Proc. Natl.Acad. Sci. U.S.A 95:2481-2485 (1998).

[0008] The pathogenesis of parvovirus infection has been most recentlyreviewed by Parish, C. R., Baillieres Clin. Haematol. 8:57-71, (1995.).Feline parvovirus (FPV) is closely related to canine parvovirus and therespective diseases are similar in pathogenesis. Parvovirus replicatesfirst in the tonsils, and then spreads to its target cells: mitoticallyactive intestinal crypt epithelial cells and bone marrow stein cells.Viremia lasts for less than 7 days before death or recovery. Clinicalsigns in cats include fever, vomiting, diarrhea, panlcukopenia, acuteshock and death. The disease outcome is proportional to the severity ofthe leukopenia; cats with severe panlcukopenia will often die, whilethose with mild leukopenia will usually survive.

[0009] The VP2 (or E2) epitope of mink enteritis virus (MEV), which isclosely related to FPV, has been previously expressed on the surface ofcowpea mosaic virus, which was propagated on the leaves of theblack-eyed bean (Dalsgaard, K et al., Nature Biotechnol. 15:248-252(1997)). One mg of the cow pea mosaic virus material that expressed thisepitope was used to immunize minks against virulent MEV. The minks wereprotected against clinical disease, and shed very little virus. Theauthors suggested that this epitope, expressed in this manner, couldalso be used to protect cats and dogs against their respectiveparvovirus infections.

[0010] The coding sequence for VP2 (E2) and the rabies spikeglycoprotein have also been engineered into raccoon poxvirus to make afive recombinant vaccine against rabies and feline panleukopenia (Hu, L.et al., 1996. Virology 218:248-252., Hu, L. et al., 1997, Vaccine 15:1466-1472.). Cats vaccinated with this construct showed excellentprotection against virulent parvovirus challenge.

[0011] Citation of the above documents is not intended as an admissionthat any of the foregoing is pertinent prior art. All statements as tothe date or representation as to the contents of these documents isbased on the information available to the applicant and does notconstitute any admission as to the correctness of the dates or contentsof these documents.

SUMMARY OF THE INVENTION

[0012] The present invention provides recombinant plant viruses thatexpress fusion proteins that are formed by fusion between a plant viralcoat protein (VCP) and a peptpide or polypeptide of interest, primarilya peptide that bears an epitope of FLV. By infecting plant cells withthe recombinant plant viruses of the invention, relatively largequantities of the peptide, in the form of a fusion protein, is produced.The fusion protein encoded by the recombinant plant virus may beengineered to have a variety of structures. The peptide may be fused tothe amino terminus (N-terminus) or to the carboxy-terminus (C-terminus)of the VCP. Alternatively, the peptide may be fused internally into acoat protein (so that it is flanked on either side by coat proteinsequences). The VCP fusion protein should have one or more properties ofthe fused peptide/polypeptide. The recombinant coat fusion protein maybe used as all immunogen or antigen to include an antibody response andprotective immunity, or as reagent for developing and conductingimmunoassays.

[0013] This invention also provides a polynucleotide that includes thegenome of a recombinant plant virus. In another aspect, the inventionprovides the coat fusion protein that is encoded by the recombinantplant virus. Yet another embodiment is a plant cell or a whole plantthat has been infected with this recombinant plant virus.

[0014] In particular, the invention provides a polynucleotide encoding afusion protein capable of being expressed in a plant or a plant cell,wherein the fusion protein comprises (a) a plant VCP from asingle-stranded plus-sense RNA virus fused to a peptide of interest,preferably comprising the amino acid sequence MGSDGAVQPDGGQPAV [SEQ IDNO:1] or comprising the amino acid sequence MGQPDGGQPAVRNERAT [SEQ IDNO:2], and (b) a promoter functional in plants that is situated 5′ tothe fusion protein coding region.

[0015] The present invention is an important step forward in the art asit provides:

[0016] 1. Stable, soluble, extractable TMV N-terminal coat proteinfusion products useful as immogens and vaccines.

[0017] 2. The first description of a polyethylencimine based virusextraction method.

[0018] 3. High yields and stabilities of protein product based upon theplant hosts that express, and extraction methods that are used topurify, the product. The interplay of the particular production host andextraction methods that maximize the amount of undegraded productcontribute to the high yields.

[0019] 4. Specific and safe immunologically active epitopes decoratingvirus particles.

[0020] The novel aspects of this invention are not limited inapplication to the disclosed parvovirus vaccines, but rather areapplicable to the highly efficient and inexpensive production of anyrelevant biologically active protein product for commercial use. Thepresent inventors have shown that TMV-based vectors can express smallantigenic peptides on the virion surface, that these virions can beeasily and highly purified from infected leafs, and that an exemplarypeptide (FPV-E2) provides the basis for a safe and efficacious vaccinerequiring no further adjuvant over and above the immunogen itself. Theinterstitial fluid of tobacco plants infected with particular TMVvectors containing the apropriate gene is safe, without requiringfurther purification, for pharmaceutical use, for example for injectioninto cats as a parvovirus vaccine.

BRIEF DESCRIPTION OF THE DRAWINGS

[0021]FIG. 1 shows the mass spectrometric data for construct 149 grownin Nicotiana benthamiana and purified by pH and heat treatment.

[0022]FIG. 2 shows the mass spectrometric data for construct 149 grownin Nicotiana tabacum and purified by pH and heat treatment.

[0023]FIG. 3 shows the mass spectrometric data for construct 150 grownin Nicotiana benthamiana and purified by PEI treatment method.

[0024]FIG. 4 shows the mass spectrometric data for construct 150 grownin Nicotiana benthamiana and purified by pH and heat treatment.

[0025]FIG. 5 shows the mass spectrometric data for construct 149 grownin Nicotiana tabacum and purified by pH and heat treatment.

[0026]FIG. 6 shows the mass spectrometric data for construct 150 grownin Nicotiana tabacum and purified by pH and heat treatment.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] The subject invention provides novel recombinant plant virusesinclude in their genetic material nucleotide sequences that encodefusion proteins that consist of a plant viral coat protein (VCP) and anadditional peptide/polypeptide of interest. The peptides of particularinterest are the feline parvovirus peptide designated E1, which has theamino acid sequence MGSDGAVQPDGGQPAV [SEQ ID NO:1], and E2 which has theamino acid sequence MGQPDGGQPAVRNERAT [SEQ ID NO:2] as well as similarpolypeptides that provide protective immunity against parvovirus whenproduced as part of a VCP fusion.

[0028] The recombinant plant viruses of the invention permit systemicexpression of the fusion protein in an infected plant. Thus by employingthese recombinant plant viruses, large quantities of a peptide ofinterest (as fusion protein) may be produced.

[0029] The fusion proteins of the invention comprise: (i) a plant VCPand (ii) a peptide or polypeptide of interest. The plant VCP portion maybe derived from the same virus from which the genome of the expressionvector is primarily derived. That is, the coat protein is native withrespect to the recombinant viral genome. Alternatively, the coat proteinfusion partner may be heterologous, that is, non-native, in that it isderived from a virus different that that which contributes therecombinant viral genome. In a preferred embodiment, the 17.5 kDa coatprotein of TMV is used in conjunction with a TMV-derived vector.

[0030] The peptide/polypeptide of interest in the protein may consist ofa peptide of virtually any amino acid sequence, provided that thepeptide does not significantly interfere with (1) the ability of thefusion protein to bind to a receptor molecule, including to antibodiesand T cell receptors; (2) the ability to bind to the active site of anenzyme (3) the ability to induce an immune response, (4) or any othebiological activity which may be required of the fusoin protein,including hormonal activity, immunoregulatory activity or metalchelating activity, just to name a few. The peptide of interest may alsopossess additional chemical or biological properties that have not beenenumerated. The peptide for fusing may may be obtained by employing allor part of the amino acid residues of a protein known to have thedesired properties. For example, an amino acid sequence of hepatitis Bsurface antigen may be used as the peptide of interest herein, yieldinga fusion protein that shares antigenic properties with hepatitis Bsurface antigen. Detailed structural and functional information aboutmany proteins are well known; this information may be used by the personof ordinary skill in the art in selecting a peptide or polypeptide forthe present fusion proteins that will have desired properties.

[0031] The peptide/polypeptide of interest may vary in size and isdefined herein as having as few as one amino acid residue to overseveral hundred amino acid residues. Preferably, the peptide in thesubject fusion protein is less than 100 amino acid residues in size,more preferably, less than 50 residues. It will be appreciated by thoseof ordinary skill in the art that in some embodiments of this invention,the peptide of interest portion may need to exceed 100 residues in orderto maintain the desired structure and properties. Preferably, the sizeof the peptide of interest in the fusion protein is minimized whenpossible provided of course that it retains the desiredbiological/chemical properties.

[0032] While this peptide may be derived from any of the variety ofproteins, a preferred protein is one against which an immune response isdesired, so that tht peptide serves as an antigen, preferably inimmunogenic form. For example, the fusion protein, or a fragmentthereof, may be injected into a mammal, along with a suitable adjuvant,to induce an immune response directed against the peptide of interest.The immune response against this peptide domain of the fusion proteinhas numerous advantages, such as protection against infection and thegeneration of antibodies useful in immunoassays.

[0033] The location (or locations) in the VCP where the peptide ofinterest is joined (fused) to the VCP is referred to herein as the“fusion joint.” A given fusion protein may have one or two fusionjoints. The fusion joint may be located at the C-terminus of the VCPwhere it is fused to the N- terminus of the peptide of interest. Thefusion joint may be located at the N-terminus of the VCP where it isfused to the C-terminus of the peptide of interest. In other embodimentsof the invention, the peptide of interest is located internally withinthe VCP; in this case, the fusion protein will have two fusion joints.This is termed an internal fusion protein. Internal fusion proteins maycomprise an entire plant VCP or a fragment thereof that is “interrupted”by the peptide of interest. The fusion joints may be located at avariety of sites within a coat protein. The entire peptide may lie inthe N-terminal portion or the C-terminal portion of the VCP. Suitablesites for the fusion joints may be determined either through routinesystematic variation, testing the resultant internal fusion protein forthe desired properties. Suitable sites for the fusion joints may also bedetermined by inspection of the three dimensional structure of the coatprotein to determine sites for “insertion” of the peptide that will notsignificantly interfere with the structural and biological functions ofthe VCP portion of the fusion protein. Detailed three dimensionalstructures of plant VCPs and their orientation in the virus have beendetermined and are publicly available to a person of ordinary skill inthe art. For example, a resolution model of the coat protein of CucumberGreen Mottle Mosaic Virus (a coat protein bearing strong structuralsimilarities to other tobamovirus coat proteins) and the virus can befound in Wang and Stubbs, J Mol. Biol. 239:371-384 (1994). Detailedstructural information of TMV can be found, among other places, in Nambaet al., J. Mol. Biol. 208:307-325 (1989) and Pattanayok and Stubbs, J.Mol. Biol. 228:516-528 (1992).

[0034] Knowledge of the three dimensional structure of a plant virusparticle and the assembly process of the virus particle permits theperson of ordinary skill in the art to design various VCP fusions of theinvention, including insertions, and partial substitutions. For example,if the peptide of interest is hydrophilic, it may be appropriate to fusethe peptide to the TMV coat protein (TMVCP) region known to be orientedas a surface loop region. Likewise, α helical segments that maintainsubunit contacts might be substituted for appropriate regions of theTMVCP helices or nucleic acid binding domains expressed in the region ofthe TMVCP oriented towards the genome.

[0035] Polynucleotide sequences encoding the subject fusion proteins maycomprise a “leaky” stop codon at a fusion joint. The stop codon may bepresent as the codon immediately adjacent to the fusion joint, or may belocated close (e.g., within 9 bases) of the codons encoding the fusionjoint. The purpose for such a leaky stop codon is to maintain a desiredratio of fusion protein to wild type coat protein. A “leaky” stop codondoes not always result in translational termination and is periodicallytranslated. The frequency of initiation or termination at a givenstart/stop codon is context dependent. The ribosome scans from the5′-end of a mRNA for the first ATG codon. If it is in a non-optimalsequence context, the ribosome will pass, at a certain frequency, to thenext available start codon and initiate translation downstream of thefirst. Similarly, the first termination codon encountered duringtranslation will not always function if it is in a particular sequencecontext. Consequently, many naturally occurring proteins exist as apopulation having heterogeneous N and/or C terminal extensions. Byincluding a leaky stop codon at a fusion joint coding region in arecombinant viral vector encoding a VCP fusion protein, the vector maybe used to produce both the longer fusion protein and a second shorterprotein, e.g. the VCP itself. A leaky stop codon may be used at, orproximal to, the fusion joints of fusion proteins in which the peptideof interest portion is joined to the C-terminus of the coat proteinregion, whereby a single recombinant viral vector could produce both VCPfusion proteins and VCPs. Additionally, a leaky start codon may be usedat or near the fusion joints to obtain a similar result. In the case ofTMVCP, extensions at the N and C-terminus are localized to the surfaceof viral particles and can be expected to project away from the helicalaxis. An example of a leaky stop sequence occurs at the junction of the126/183 kDa reading frames of TMV as was described years ago (Pelham, H.R. B., Nature 272:469-471 (1978). Skuzeski, J. M. et al., J Mol. Biol.20:365-373 (1991), defined necessary 3′ context requirements of thisregion to confer leakiness of termination on a heterologous proteinmarker gene (β-glucuronidase) as CAR-YYA (R=purine; Y=pyrimidine).

[0036] In another embodiment of the invention, the fusion joints on thefusion proteins are designed to be cleavable by having an amino acidsequence that is a substrate for a protease. This permits separation andisolation of the peptide of interest by using a suitable proteolyticenzyme. The proteolytic enzyme may contact the fusion protein either invitro or in vivo.

[0037] The expression of the fusion protein may be driven by any of avariety of promoters functional in the context of the recombinant plantviral vector and host plant. In a preferred embodiment plant viralsubgenomic promoters are used (U.S. Pat. No. 5,316,931).

[0038] Recombinant DNA technologies have allowed the life cycle ofnumerous plant RNA viruses to be extended artificially through a DNAphase that facilitates manipulation of the viral genome. Thesetechniques may be applied by the person of ordinary skill in the art inorder make and use recombinant plant viruses of the invention. Theentire cDNA of the TMV genome was cloned and functionally joined to abacterial promoter in an E. coli plasmid (Dawson, W. O. et al., Proc.Natl. Acad. Sci. U.S.A 83:1832-1836 (1986)). Infectious recombinantplant viral RNA transcripts may also be produced using other well knowntechniques, for example, commercially available RNA polymerases from T7,T3 or SP6. Precise replicas of the virion RNA can be produced in vitrowith RNA polymerase and dinucleotide cap, m7GpppG. This not only allowsmanipulation of the viral genome for reverse genetics, but it alsoallows manipulation of the virus into a vector to express foreign genes.A method of producing plant RNA virus vectors based on manipulating RNAfragments with RNA ligase has proved to be impractical and is not widelyused (Pelcher, L. E. et al., EP 67553A2 (1982). Detailed information onhow to make and use recombinant RNA plant viruses can be found, amongother places in U.S. Pat. No. 5,316,931 (Donson et al.), which is hereinincorporated by reference. The invention provides nucleic acids thatcomprise a recombinant RNA plant vector for expression of the subjectfusion proteins. The invention also provides for nucleic acids thatcomprise a portion or portions of the subject vectors. The vectorsdescribed in U.S. Pat. No. 5,316,931 are particularly preferred forexpressing the fusion proteins of the invention.

[0039] This invention also provides virus particles that comprise thesubject fusion proteins. The coat of the virus particles of theinvention may consist entirely of VCP fusion protein. In anotherembodiment, the virus particle coat consists of a mixture of VCP fusionproteins and non-fused VCP, wherein the ratio of the two proteins mayvary. As tobamovirus coat proteins may self-assemble into virusparticles, the virus particles of the invention may be assembled eitherin vivo or in vitro. The virus particles may also be convenientlydisassembled-using well known techniques so as to simplify thepurification of the subject fusion proteins, or portions thereof.

[0040] The invention also provides recombinant plant cells comprisingthe subject fusion proteins and/or virus particles comprising thesubject fusion proteins. These plant cells may be produced either byinfecting plant cells (in culture or in whole plants) with theinfectious recombinant virus particles of the invention or withpolynucleotides comprising the genomes of the infectious virus particleof the invention. The recombinant plant cells of the invention have manyuses, chief among which is serving as a source for the fusion coatproteins of the invention.

[0041] The peptide portion of the subject fusion proteins may comprisemany different amino acid sequences, and accordingly may have differentbiological/chemical properties. In a preferred embodiment, the peptideportion of the fusion protein is a vaccine antigen. The surface of TMVparticles and other tobamoviruses contain continuous epitopes of highantigenicity and segmental mobility thereby making TMV particlesespecially useful in inducing a desired immune response. Theseproperties make the virus particles of the invention especially usefulas carriers of foreign epitopes to mammalian immune systems.

[0042] While the recombinant RNA viruses of the invention may expressnumerous coat fusion proteins for use as vaccine antigens (or theirprecursors), an important embodiment is a vaccine composition againstmalaria. Human malaria is caused by the protozoan species Plasmodiumfalciparum, P. vivax, P. ovale and P. malariae and is transmitted in thesporozoite form by the Anopheles mosquito. Control of this disease willlikely require safe and stable vaccines. Several peptide epitopesexpressed during various stages of the parasite life cycle are thoughtto contribute to the induction of protective immunity in partiallyresistant individuals living in endemic areas and in individualsexperimentally immunized with irradiated sporozoites.

[0043] When the fusion proteins of the invention, fragments thereof orviral particles expressing the proteins or fragments are to beadministered in vivo, they are typically given as a pharmaceuticalcomposition that includes a pharmaceutically acceptable carrier orexcipient. Such as carrier can be any compatible, non-toxic substancesuitable for delivery of the desired compounds to the body. Sterilewater, alcohol, fats, waxes and inert solids may be included in thecarrier. Pharmaceutically accepted buffering agents, dispersing agents,etc. may also be incorporated into the pharmaceutical composition.Additionally, when fusion proteins or fragments are used to induceimmune responses (protective or otherwise), the formulation may compriseone or more immunological adjuvants in order to stimulate a more potentdesired immune response.

[0044] Any of a number of routes of administration may be used whengiving the compositions to an animal, including a human. Thepharmaceutical compositions may be administered orally or parenterally,i.e.,, subcutaneously, intradermally, intramuscularly or intravenously.Compositions for parenteral administration comprise a solution of thefusion protein (or derivative) or a cocktail thereof dissolved in anacceptable carrier, preferably an aqueous carrier, e.g., water, bufferedwater, 0.4% saline, buffered saline, 0.3% glycerine and the like. Thesesolutions are sterile and generally free of particulate matter. Thesecompositions may be sterilized by conventional sterilization techniques.The compositions may contain pharmaceutically acceptable auxiliarysubstances as required to approximate physiological conditions such aspH adjusting and buffering agents, toxicity adjusting agents and thelike, for example sodium acetate, sodium chloride, potassium chloride,calcium chloride, sodium lactate, etc.

[0045] The concentration of fusion protein (or portion thereof) in theseformulations can vary widely depending on the specific amino acidsequence and the desired biological activity, e.g., from less than about0.5%, usually at least about 1% to as much as 15 or 20% by weight andwill be selected primarily based on fluid volumes, viscosities, etc., inaccordance with the particular mode of administration selected and thecondition of the recipient.

[0046] Actual methods for preparing parenterally administrablecompositions and adjustments necessary for administration to subjectsare known or apparent to those skilled in the art and are described inmore detail in, for example, Remington's Pharmaceutical Science, currentedition, Mack Publishing Company, Easton, Pa., which is incorporatedherein by reference.

[0047] Having now generally described the invention, the same will bemore readily understood through reference to the following exampleswhich are provided by way of illustration, and are not intended to belimiting of the present invention, unless specified.

EXAMPLES

[0048] The following present examples are based on a full length insertof wild type TMV (U1 strain) cloned in the vector pUC 18 with a T7promoter sequence at the 5═-end and a KpnI site at the 3′-end (pSNC004,FIG. 2) or a similar plasmid pTMV304. Using the polymerase chainreaction (PCR) technique and primers WD29 (SEQ ID NO:3) and D1094 (SEQID NO: ), a 277 XmaI/HindIII amplification product was inserted with the6140 bp XmaI/KpnI fragment from pTMV304 between the KpnI and HindIIIsites of the common cloning vector pUC18 to create pSNC004. The plasmidpTMV304 is available from the American Type Culture Collection,Rockville, Md. (ATCC Accession # 45138). The genome of the wild type TMVstrain can be synthesized from pTMV304 using the SP6 polymerase, or frompSNC004 using the T7 polymerase. The wild type TMV strain can also beobtained from the American Type Culture Collection, Rockville, Md. (ATCCAccession No. PV135). The plasmid pBGC152, Kumagai, M., et al. (1993),is a derivative of pTMV304 and is used only as a cloning intermediate inthe examples described below. The construction of each plasmid vectordescribed in the examples below is diagrammed in FIG. 3.

Example 1 Construction of pJL 60.3

[0049] To facilitate cloning of TMV U1 CP fusions into an infectious TMVU1 cDNA backbone, the vector pJL 60.3 was constructed. The plasmid pJL60.3 contains a full length infectious clone of TMV U1 with a smallmultiple cloning site polylinker:

[0050] taaatattcttaagccagtagtatgggatatccagtggtatgggatcctacagtatc [SEQ IDNO:5] containing two BstXI sites, CCAGTAGTATGG [SEQ ID NO:6] andCCAGTGGTATGG [SEQ ID NO:7), separated by a unique EcoRV site (GATATC),between the stop codon of the 30K protein gene and the start codon ofthe U1 CP.

[0051] To construct pJL 60.3, a 0.7 kb DNA fragment comprising the TMVU1 CP and 3′ UTS was PCR amplified from pBTI 801 using the followingprimers:

[0052] Kinased 5′ Primer JAL 72

[0053] tgggatatccagtggtatgggatcctacagtatacactactccatctcag [SEQ ID NO: 8]and

[0054] 3′ primer JON 56

[0055] cgcqtacctgggcccctaccgggggtaacg [SEQ ID NO:9] pBTI 801 contains afull length infectious clone TMV U1, under the control of the T7promoter sequence, in a pUC based plasmid. A KpnI restriction enzymesite lies at the 3′ end of the viral cDNA, immediately followed by aself-processing ribozyme sequence from sattelite tobacco ringspot virusRNA. The presence of this self-processing ribozyme downstream of the TMV3′ end allows for the transcription of the TMV cDNA without priorlinearization of the plasmid template DNA (with KpnI, for example)).

[0056] A 0.3 kb fragment of pBTI 801 was then PCR amplified using thefollowing primers:

[0057] 5′ Primer JON 52 (TMV U1 nts 5456-5482):

[0058] ggcccatggaacttacagaagaagtcg [SEQ ID NO:10]

[0059] Kinased 3′ Primer JAL 73

[0060] ctggatatcccatactactggcttaagaatatttaaaacgaatccgattcggcgaca [SEQ IDNO:11]

[0061] The 0.7 kb PCR product, containing the EcoRV and BstXI siteCCAGTGGTATGG [SEQ ID NO:7] upstream of the U1 CP ORF and 3′ UTS, wasthen ligated to the 0.3 bp PCR products (which contained the 3′ end ofthe TMV 30K protein gene and the BstXI site CCAGTAGTATGG [SEQ ID NO:6]downstream of the 30K protein stop codon. The product of this ligationreaction was then used in a PCR with 5′ primer JON 52 (shown above) 3′primer JON56 (shown above). to generate a 1 kb PCR product. That productwas digested with PacI and NcoI, and the digested DNA waselectrophoresed on an agarose gel. The NcoI site is contained within theprimer sequence of JON 52, and the PacI site is a unique restrictionsite in the TMV U1 CP gene sequence. The 0.4 kb PacI-NcoI fragment wasthen isolated from an agarose gel and ligated into a PacI-NcoI digested8.8 kb fragment of pBTI 801 to generate pJL 60.3.

[0062] Again, the relevant feature of pJL 60.3 for the construction ofpBTI 2149 and pBTI 2150 is the existence of the BstXI site CCAGTAGTATGG[SEQ ID NO:6] between the TMV 30 K stop codon and the CP start codon.

Example 2 Construction of Plasmid pBTI 2149

[0063] A 0.7 kb DNA fragment comprising the TMV U1 coat protein (CP) and3′ UTS was PCR amplified from p BTI 801 using the following primers:5′ primer JAL 149 cctgggccagtagtatgggttcagatggtgctgtac [SEQ ID NO:12]aaccagatggaggtcaaccagctgtatcttacagta tcactactccatctcagtt

[0064] 3′ Primer JON 56 (Shown Above)

[0065] JAL 149 contains the BstXI restriction enzyme site (underscored)for cloning purposes and the coding sequence for the parovirus epitopeMGSDGAVQPDGGQPAV [SEQ ID NO:1] and TMV U1 nts 5715-5743). The amplifiedproduct comprising the parvovirus epitope fused to the U1 CP gene wasdigested with KpnI and BstXI and ligated into the 8.4 kb KpnI-BstXIfragment of pJL 60.3 to generate pBTI 2149.

[0066] Plasmid vectors pBTI 2149 encodes the recombinant virus having afusion protein of MGSDGAVQPDGGQPAV [SEQ ID NO:1] fused to the N-terminusof the coat protein. Plasmid vectors pBTI 2149 was deposited at the ATCCon Feb. 17, 2000, under the Budapest Treaty. The deposit bears the ATCCaccession #PTA-1403.

Example 3 Construction of Plasmid pBTI 2150

[0067] A 0.7 kb DNA fragment comprising the TMV U1 coat protein (CP) and3′ UTS was PCR amplified from p801 (basically pTMV 204) using thefollowing primers: 5′primer JAL 150 cctgggccagtagtatgggttcagatggtgctgtac[SEQ ID NO:13] aaccagatqgaggtcaaccagctgtatcttacagta tcactactccatctcagtt

[0068] 3′ Primer JON 56. (Shown Above)

[0069] (The “forward” primer JAL 150 contains a BstXI restriction enzymesite (underscored above) for cloning purposes, the coding sequence forthe parovirus epitope MGQPDGGQPAVRNERAT [SEQ ID NO:2 ] and TMV U1 nts5718-5743.) The amplified product comprising the parvovirus epitopefused to the U1 CP gene was digested with KpnI and BstXI and ligatedinto the 8.4 kb KpnI-BstXI fragment of pJL 60.3 to generate pBTI 2150.

[0070] Plasmid vectors pBTI 2150 encodes the recombinant virus having afusion protein of MGQPDGGQPAVRNERAT [SEQ ID NO:2] fused to theN-terminus of the coat protein. Plasmid vectors pBTI 2150 was depositedat the ATCC on Feb. 17, 2000, under the Budapest Treaty. The depositbears the ATCC accession # PTA-1404.

Example 4 Production of Virus TMV 149

[0071] The virus TMV 149 was produced by transcription of plasmid pBTI2149. Infectious transcripts were synthesized from transcriptionreactions with T7 RNA polymerase in the presence of cap analog (7mcpppG)(New England Biolabs) according to the manufacturers instructions.Transcripts were used to inoculate N. benthamiana leaves which had beenlightly dusted with carborundum (silicon carbide 400 mesh, Aldrich).

Example 5 Production of Virus TMV 150

[0072] The virus TMV 150 was produced by transcription of plasmid pBTI2150. Infectious transcripts were synthesized from transcriptionreactions with T7 RNA polymerase in the presence of cap analog (7mGpppG)(New England Biolabs) according to the manufacturers instructions.Transcripts were used to inoculate N. benthamiana leaves which had beenlightly dusted with carborundum (silicon carbide 400 mesh, Aldrich).

Example 6 Extraction and Purification of TMV Coat Protein Fusion Virions

[0073] The two TMV coat fusion constructs were expressed in andextracted from Nicotiana benthamiana and/or Nicotiana tabacum using apH-heat or PEI extraction method as described below, and in Table 1.Virus preparations were characterized using Matrix Assisted LaserDesorption Ionization—Time of Flight (MALDI-TOF) (Example 7; see alsoTable 2). Based upon the product masses determined by MALDI andpolyacrylamide gel electrophoresis (PAGE) analysis, a proteolyticdegradation profile was determined for each construct for any given hostplant or extraction method used to produce the coat fusion product.(Sees Tables 2 and 3).

[0074] A. pH-Heat Extraction

[0075]Nicotiana benthamiana or Nicotiana tabacum cv MD609, produced in agrowth rooms, were inoculated with TMV derivatives containing parvovirusepitopes fused to the Nterminus of the coat protein. Plants wereharvested 2.5-5 weeks post inoculation after systemic spread of thevirus. Leaf and stalk tissue (150 g) was macerated in a 1 L Waringblender for 2.0 minutes at the high setting with 300 ml of chilled,0.04% Na₂S₂O₅. The macerated material was strained through four layersof cheesecloth to remove fibrous material. The resultant “green juice”was adjusted to a pH of 5.0 with H₃PO₄. The pH adjusted green juice washeated to 47° C. and held at this temperature for 5 minutes and thencooled to 15° C. The heat-treated green juice was centrifuged at 6,000×Gfor 3 minutes resulting in two fractions, supernatant 1 and pellet 1.The pellet 1 fraction was resuspended in distilled water using a volumeof water equivalent to 1/z of the initial green juice volume. Theresuspended pellet 1 was adjusted to a pH of 7.5 with NaOH andcentrifuged at 6,000×G for 3 minutes resulting in two fractions,supernatant 2 and pellet 2. Virus was precipitated from both supernatantfractions 1 and 2 by the addition of polyethylene glycol (PEG) 6,000 andNaCl (4% by volume). After incubation at 4° C. (1 hour), precipitatedvirus was recovered by centrifugation at 10,000×G for 10 minutes. Thevirus pellet was resuspended in 10 mM NaKPO₄ buffer, pH 7.2 andclarified by centrifugation at 10,000×G for 3 minutes. The clarifiedvirus preparation was precipitated a second time by the addition ofpolyethylene glycol (PEG) 6,000 and NaCl (4% by volume). Precipitatedvirus was recovered by centrifugation as described above. Virus yieldsare shown in Table 1.

[0076] B. Polyethyleneimine (PEI) Extraction

[0077]Nicotiana benthamiana or Nicotiana tabacum cv MD609, produced in agrowth rooms, were inoculated with tobacco mosaic virus derivativescontaining parvovirus epitopes fused to the Nterminus of the coatprotein. Plants were harvested 2.5-5 weeks post inoculation aftersystemic spread of the virus. Leaf and stalk tissue (150 g) wasmacerated in a 1 L Waring blender for 2.0 minutes at the high settingwith 300 ml of chilled, 50 mM Tris, pH 7.5, 2 mM EDTA and 0. 1%β-mercapto-ethanol. The macerated material was strained through fourlayers of cheesecloth to remove fibrous material. The resultant “greenjuice” was adjusted to 0.1% polyethylenimine, PEI (Sigma, St. Louis,Mo.) by the addition of a 10% PEI WNV stock solution. The PEI treatedgreen juice was stirred for 30 minutes, (4° C.) and then centrifuged at3,000×G for 5 minutes resulting in two fractions, supernatant 1 andpellet 1. The pellet 1 fraction was resuspended in distilled water usinga volume of water equivalent to ½ of the initial green juice volume. Theresuspended pellet 1 was adjusted to a pH of 7.5 with NaOH andcentrifuged at 6,000×G for 3 minutes resulting in two fractions,supernatant 2 and pellet 2. Virus was precipitated from both supernatantfractions 1 and 2 by the addition of polyethylene glycol (PEG) 6,000 andNaCl (4% by volume). After incubation at 4° C. (1 hour), precipitatedvirus was recovered by centrifugation at 10,000×G for 10 minutes. Thevirus pellet was resuspended in 10 mM NaKPO₄ buffer, pH 7.2 andclarified by centrifugation at 10,000×G for 3 minutes. The clarifiedvirus preparation was precipitated a second time by the addition ofpolyethylene glycol (PEG) 6,000 and NaCl (4% by volume). Precipitatedvirus was recovered by centrifugation as described above. Virus yieldsare shown in Table 1. TABLE 1 Virus Yield Vector Host Plant ExtractionMethod Virus Yield* TMV149 N. benthamiana PH-Heat, Supernatant 1 0.3929TMV149 N. benthamiana PH-Heat, Supernatant 2 0.0396 TMV149 N.benthamiana PEI, Supernatant 1 0.0005 TMV149 N. benthamiana PEI,Supernatant 2 — TMV149 N. tabacum PH-Heat, Supernatant 1 0.0488 TMV149N. tabacum PH-Heat, Supernatant 2 0.0376 TMV149 N. tabacum PEI,Supernatant 1 — TMV149 N. tabacum PEI, Supernatant 2 — TMV150 N.benthamiana PH-Heat, Supernatant 1 1.2274 TMV150 N. benthamiana PEI,Supernatant 2 0.8860 TMV150 N. benthamiana PEI, Supernatant 1 1.5369TMV150 N. tabacum PEI, Supernatant 2 — TMV150 N. tabacum PH-Heat,Supernatant 1 0.321 TMV150 N. tabacum PEI, Supernatant 1 0.0368 TMV150N. tabacum PEI, Supernatant 2 0.0001

[0078] The yield of epitope specific virus particles is dependent uponthe species of plant used as the virus host and method of extraction.TMV149 yielded the highest quantity of virus when produced in N.benthamiana and extracted using the pH-heat method. In addition, theTMV149 particles partitioned primarily into supernatantl. Negligibleyields of TMV149 were observed when the PEI method was employed. TMV150yielded the highest quantity of virus when produced in N. benthamianaand extracted using the PEI method. TMV150 partitioned into bothsupernatant 1 and 2 (60% and 40%, respectively) when extracted by thepH-heat method.

Example 7 Analysis of Coat Protein Fusions by MALDI

[0079] PEG precipitated, resuspended virus preparations were diluted in50% acetonitrile and further diluted 1:1 with sinapinic acid (Aldrich,Milwaukee, Wis.). The sinapinic acid was prepared at a concentration of10 mg/ml in 0.1% aqueous triflouroacetic acid/acetonitrile (70/30 byvolume). The sinapinic acid treated sample (1.0 μl) was applied to astainless steel MALDI plate surface and allowed to air dry at roomtemperature. MALDI-TOF mass spectra were obtained with a PerSeptiveBiosystems DE-PRO (Houston, Tex.) operated in the linear mode. A pulsedlaser operating at 337 rim was used in the delayed extraction mode forionization. An acceleration voltage of 25 kV with a 90% grid voltage anda 0.1% guide wire voltage was used. Approximately 100 scans wereacquired and averaged over the mass range 2,000-156,000 Da with a lowmass gate of 2,000. Ion source and mirror pressures were approximately1.2×10⁻⁷ and 1.6×10⁻⁷ Torr, respectively. All spectra were masscalibrated with a single-point fit using horse apomyoglobin (16,952 Da).TABLE 2 Product Mass Characterization Days Extraction Post Method Inocu-and Product Mass Plant Host/Vector lation Fraction (MALDI) Daltons*, **N. benthamiana/ 17 PH-Heat 18,822 (50%); 18,766 (50%)** TMV149 Super-natant 1 N. tabacum/ 17 PH-Heat 18,823 (40%); 18,762 (40%) TMV149 Super-18,564 (<2%); 18,509 (<2%); natant 1 18,442 (2%); 18,329 (<2%); 17,993(10%); 17,935 (2%) N. tabacum/ 35 PH-Heat, 18,812 (60%); 18,752 (40%)TMV149 super- natant 1 N. benthamiana/ 17 PH-Heat, 19,025 (>95%); 17,964(<5%) TMV150 super- natant 1 N. benthamiana/ 17 PEI, 19,029 (>95%);17,980 (<5%) TMV150 Super- natant 1 N. tabacum/ 17 pH-Heat, 19,020(60%); 17,956 (40%)** TMV150 Super- natant 1 N. tabacum/ 35 pH-Heat,19,020 (80%); 17,956 (20%) TMV150 Super- natant 1 N. tabacum/ 17 PEI,19,021 (>95%); 17,957 (<5%) TMV150 Super- natant 1

[0080] TABLE 3 Proteolytic Degradation Profiles MW (daltons) TMVI49GSDGAVQPDGGQPAVSYSITTPSQ 18,816.5 SDGAVQPDGGQPAVSYSITTPSQ 18,759.5GAVQPDGGQPAVSYSITTPSQ 18,557.4 AVQPDGGQPAVSYSITTPSQ 18,500.4VQPDGGQPAVSYSITTPSQ 18,429.4 QPDGGQPAVSYSITTPSQ 18,330.3 GGQPAVSYSITTPSQ17,990.2 GQPAVSYSITTPSQ 17,933.1 TMV150 GQPDGGQPAVRNERATYSITTPSQ19,027.7 NERATYSITTPSQ 17,965.1

[0081] The results presented in Tables 2 and 3 indicate effects of hostspecies, extraction method and extraction timing on the proteolysis ofN-terminal TMV coat protein fusions. In all cases, the terminal Metresidue is removed from all fusions, as is the case with native coatprotein. The N-terminal glycine residue is removed from 40-60% of theTMV149 fusions. Extractions (pH-heat) performed on TMV149 and 150produced in 17 day post inoculated N. tabacum, resulted in the mostcomplex and greatest degree of proteolytic activity. The differences inproteolytic degradation may reflect both qualitative and quantitativedifferences in proteases present in different plant species or atdifferent plant development periods. The PEI extraction of TMV150 provedto be protective, resulting in negligible degradation relative to thepH-heat extraction (N. tabacum host).

Example 8 Virion Purification and Formulation for Use in Animal Studies

[0082] PEG precipitated virion preparations (see Table 4) wereresuspended in water for injection (WFI) at a concentration of 1.0 mgvirus per 1.0 ml WFI. All laboratory ware used to process the viruspreparations was baked at 225° C. for 18 hours. The resuspended viruspreparation was solvent-extracted with chloroform and 1-butanol (8% byvolume) by intermittent shaking for 1 hour at room temperature. Phaseswere separated by centrifugation at 10,000×G for 5 minutes. The aqueousphase was frozen in a dry ice/methanol bath and lyophilized overnightuntil dry. The lyophilized virus preparation was resuspended at aconcentration of 5-10 mg virus per 1.0 ml WFI. The resuspended viruspreparation was packaged in 10 ml serum vials that were sealed bycrimping. TABLE 4 TMV Fusions Preparations Processed for Animal StudiesTMV Fusion Host Extraction Method TMV149 N. benthamiana PH-Heat,Supernatant 1 TMV150 N. benthamiana PEI, Supernatant 1

[0083] Samples selection for further processing was based on both yieldand percentage of fusion that remained undegraded (based on MALDIanalysis).

Example 9 Vaccine Testing

[0084] The parvovirus vaccine, utilizing tobacco plant expressedconstruct E1 and construct E2, was tested in young cats for safety andefficacy. E2 expressed on TMV particles proved to be safe andimmunogenic by itself. E1 vaccine was somewhat less immunogenic. Catsvaccinated with E2, E1 or a mixture of E2 and E1 all showed significantprotection against a 30% lethal dose of virulent FPV. No adjuvant wasrequired other than what was provided by TMV proteins, some of which areknown to act as superantigens (nonspecific immunostimulators). With thedevelopment and testing of this particular vaccine, the presentinventors have established the usefulness and advantages of theexpression system for producing common feline vaccines.

[0085] The E1 and E2 epitopes are the two principal hemagglutinating andneutralizing antibody-inducing antigens on the surface of FPV. Thesequences of the two epitopes overlap. Cats immunized with theseepitopes will develop virus neutralizing antibodies and will bepartially protected against challenge with virulent virus. Therefore,cats were immunized with either E1 or E2 peptides, or with both, andthen monitored for the vaccine's safety, immunogenicity and efficacy.

[0086] Cats were immunized with 100-200 μg of each peptide, starting at8-12 weeks of age, and with a second immunization 4 weeks later. Theywere then challenged orally with a large dose of virulent FPV. Bothimmunogens appeared completely safe, inducing no fever, depression orlocal reactions. Antibodies were measured using the enzyme-linkedimmunosorbent assay (ELISA). After the second immunization, significanttiters of antibodies were detected in ELISA run against wholeparvovirus. Cats receiving E1+E2 gave slightly higher responses thancats immunized with E1 or E2. After challenge, cats immunized with E2(either alone or in combination with E1) appeared to be solidlyprotected, as evidenced by minimal signs of disease and no mortality,when compared to control cats immunized with TMV alone (that did notexpress E2 or E1). It was concluded that the E2 peptide, when deliveredon TMV particles was a safe and effective vaccine, and moreover, did notrequire additional adjuvants.

[0087] To summarize:

[0088] 1. Cats immunized with E1-TMV or E2-TMV (100-200gg) madedetectable antibody responses as measured by ELISA against whole felinepanleukopenia virus (FPV).

[0089] 2. The antibodyh response to 200 μg of E1-TMV or E2-TMV wasgreater than to 100 μg.

[0090] 3. Cats immunized with a combination of E1-TMV and E2-TMV madebetter antibody responses than cats immunized with either protein alone.

[0091] 4. Cats vaccinated with E2-TMV or E1-TMV+E2-TMV showed betterprotection to virulent parvovirus challenge than control cats that wereunimmunized or immunized with TMV. E2-TMV was more protective thanE1-TMV

[0092] 5. Both E1-TMV and E2-TMV prevented mortality; E2-TMV was moreeffective at reducing morbidity. E2-TMV-immunized cats weresignificantly less febrile, showed few clinical signs of illness andwere markely less leukopenic than unimmunized cats or or cats immunizedwith control TMV.

[0093] 6. Immunity conferred by E2-TMV was not sterilizing, which istypical of killed parvovirus vaccines. Immunized cats showed mild signsof disease but had pronounced immunological memory.

What is claimed is:
 1. A polynucleotide encoding a fusion proteincapable of being expressed in a plant or a plant cell, comprising: (a) acoding region that encodes said fusion protein and includes: i. a plantviral coat protein from a single-stranded plus-sense RNA virus, andfused thereto, ii. a peptide comprising amino acids MGSDGAVQPDGGQPAV(SEQ ID NO:1) or a fragment thereof; and (b) a promoter functional inplants that is 5′ to the coding region.
 2. A polynucleotide according toclaim 1, wherein the peptide is fused to the N-terminus of the plantviral coat protein.
 3. A polynucleotide according to claim 1, whereinthe peptide is fused to the C-terminus of the plant viral coat protein.4. A polynucleotide according to claim 1, wherein said fusion protein isan internal fusion protein with respect to the coat protein.
 5. Apolynucleotide according to claim 1, further comprising (c) a fusionjoint having a leaky stop codon from a single-stranded plus-sense RNAvirus.
 6. A polynucleotide according to claim 1, wherein the peptideMGSDGAVQPDGGQPAV or fragment comprises an antigen.
 7. A polynucleotideaccording to claim 1, wherein the coat protein is a tobacco mosaic viruscoat protein.
 8. A polynucleotide according to claim 1 wherein the coatprotein is a tobamovirus coat protein.
 9. A recombinant plant viralgenome comprising a polynucleotide according to claim
 1. 10. Arecombinant plant virus particle, comprising a genome according to claim9.
 11. A recombinant plant virus having a coat protein encoded by apolynucleotide according to claim
 1. 12. A plant cell comprising apolynucleotide according to claim
 1. 13. A plant cell comprising arecombinant plant viral genome according to claim
 9. 14. A plant cellcomprising a recombinant plant virus particle according to claim
 10. 15.A plant cell comprising a recombinant plant virus according to claim 11.16. A plant comprising a polynucleotide according to claim
 1. 17. Aplant comprising a recombinant plant viral genome according to claim 9.18. A plant comprising a recombinant plant virus particle according toclaim
 10. 19. A plant comprising a recombinant plant virus according toclaim
 11. 20. A polynucleotide encoding a fusion protein capable ofbeing expressed in a plant or a plant cell, comprising: (a) a codingregion that encodes said fusion protein and includes: i. a plant viralcoat protein from a single-stranded plus-sense RNA virus, and fusedthereto, ii. a peptide comprising amino acids MGQPDGGQPAVRNERAT (SEQ IDNO:2) or a fragment thereof; and (b) a promoter functional in plantsthat is 5′ to the coding region.
 21. A polynucleotide according to claim20, wherein the peptide is fused to the N-terminus of the plant viralcoat protein.
 22. A polynucleotide according to claim 20, wherein thepeptide is fused to the C-terminus of the plant viral coat protein. 23.A polynucleotide according to claim 20, wherein said fusion protein isan internal fusion protein with respect to the coat protein.
 24. Apolynucleotide according to claim 20, further comprising (c) a fusionjoint having a leaky stop codon from a single-stranded plus-sense RNAvirus.
 25. A polynucleotide according to claim 20, wherein the peptideMGQPDGGQPAVRNERAT or a fragment comprises an antigen.
 26. Apolynucleotide according to claim 20, wherein the coat protein is atobacco mosaic virus coat protein.
 27. A polynucleotide according toclaim 20 wherein the coat protein is a tobamovirus coat protein.
 28. Arecombinant plant viral genome comprising a polynucleotide according toclaim
 20. 29. A recombinant plant virus particle, comprising a genomeaccording to claim
 28. 30. A recombinant plant virus, wherein the coatprotein is encoded by a polynucleotide according to claim
 20. 31. Aplant cell comprising a polynucleotide according to claim
 20. 32. Aplant cell comprising a recombinant plant viral genome according toclaim
 28. 33. A plant cell comprising a recombinant plant virus particleaccording to claim
 29. 34. A plant cell comprising a recombinant plantvirus according to claim
 30. 35. A plant comprising a polynucleotideaccording to claim
 20. 36. A plant comprising a recombinant plant viralgenome according to claim
 28. 37. A plant comprising a recombinant plantvirus particle according to claim
 29. 38. A plant comprising arecombinant plant virus according to claim
 30. 39. An immunochemicalreagent comprising a fusion protein capable of being produced in a plantor a plant cell, wherein the fusion protein comprises (i) a plant viralcoat protein from a single-stranded plus-sense RNA virus; and (ii) apeptide comprising amino acids MGQPDGGQPAVRNERAT or a fragment thereoffused to the N-terminus of the coat protein.
 40. A vaccine for theprotection of mammals against parvovirus comprising the immunochemicalreagent of claim
 39. 41. A vaccine according to claim 40 together with apharmaceutically or veterinarially acceptable carrier or excipient. 42.An immunochemical reagent comprising a recombinant plant virus, whereinat least one capsid of the coat protein is a fusion protein capable ofbeing produced in a plant or a plant cell, wherein the fusion proteincomprises (i) a plant viral coat protein from a single-strandedplus-sense RNA virus; and (ii) a peptide comprising amino acidsMGSDGAVQPDGGQPAV or a fragment thereof fused to the N-terminus of thecoat protein.
 43. A vaccine for the protection of mammals againstparvovirus comprising an immunochemical reagent according to claim 42.44. A vaccine according to claim 43 together with a pharmaceutically orveterinarially acceptable carrier.
 45. A vaccine according to claim 40,wherein the fusion protein is in a recombinant plant virus.
 46. Avaccine according to claim 45 wherein said virus is a live virus.
 47. Avaccine according to claim 43, wherein the recombinant plant virus is alive virus.
 48. A vaccine composition comprising a live recombinantplant virus according to claim 46 and a pharmaceutically orveterinarially acceptable carrier or excipient.
 49. A vaccinecomposition comprising a live recombinant plant virus according to claim47 and a pharmaceutically or veterinarially acceptable carrier orexcipient.
 50. A method of making the polynucleotide of claim 1,comprising ligating an oligonucleotide encoding a peptide having thesequence MGSDGAVQPDGGQPAV or a fragment thereof to a viral coat proteingene.
 51. A method of making a recombinant plant viral genome comprising(a) inserting an oligonucleotide encoding a peptide having the sequenceMGSDGAVQPDGGQPAV or a fragment thereof into the genome of asingle-stranded plus-sense RNA virus so that said oligonucleotide isfused in frame with a plant viral coat protein gene and under thecontrol of a promoter functional in plants; or (b) ligating thepolynucleotide of claim 1 to the genome of a single-stranded plus-senseRNA virus, thereby making said recombinant plant viral genome.
 52. Amethod of making the recombinant plant virus that encodes a fusionprotein that includes a plant viral coat protein from a single-strandedplus-sense RNA virus and a peptide comprising amino acidsMGSDGAVQPDGGQPAV or a fragment thereof, comprising the steps of: (a)ligating DNA encoding the fusion protein which protein is capable ofbeing expressed in a plant or a plant cell, to a DNA copy of the genomeof a single-stranded plussense RNA virus; (b) transcribing said ligatedDNA to RNA; and (c) infecting a host plant or plant cell with saidtranscribed RNA, so that said plant or plant cell makes said recombinantplant virus.
 53. A method of making the recombinant plant virus thatencodes a fusion protein that includes a plant viral coat protein from asingle-stranded plus-sense RNA virus and a peptide comprising aminoacids MGSDGAVQPDGGQPAV or a fragment thereof, comprising the steps of;(a) ligating DNA encoding the fusion protein which protein is capable ofbeing expressed in a plant or a plant cell, to a DNA copy of the genomeof a single-stranded plussense RNA virus, under the control of apromoter that is functional in plants; and (b) transforming ortransfecting a host plant or plant cell with said ligated DNA, so thatsaid DNA is expressed in said plant or plant cell, whereby said plant orplant cell makes said recombinant plant virus.
 54. A method of making aplant cell that produces a fusion protein that includes a plant viralcoat protein from a single-stranded plus-sense RNA virus and a peptidecomprising amino acids MGSDGAVQPDGGQPAV or a fragment thereof,comprising transforming or transfecting a host plant cell with the plantviral genome of claim
 51. 55. A method of making a plant cell thatproduces a fusion protein that includes a plant viral coat protein froma single-stranded plus-sense RNA virus and a peptide comprising aminoacids MGSDGAVQPDGGQPAV or a fragment thereof, comprising infecting ahost plant cell with the plant virus of claim
 52. 56. A method of makinga plant cell that produces a fusion protein that includes a plant viralcoat protein from a single-stranded plus-sense RNA virus and a peptidecomprising amino acids MGSDGAVQPDGGQPAV or a fragment thereof,comprising infecting a host plant cell with the plant virus of claim 53.57. A method of making a plant that produces a fusion protein thatincludes a plant viral coat protein from a single-stranded plus-senseRNA virus and a peptide comprising amino acids MGSDGAVQPDGGQPAV or afragment thereof, comprising transforming or transfecting a host plantwith the plant viral genome of claim
 51. 58. A method of making a plantthat produces a fusion protein that includes a plant viral coat proteinfrom a single-stranded plus-sense RNA virus and a peptide comprisingamino acids MGSDGAVQPDGGQPAV or a fragment thereof, comprising infectinga host plant with the plant virus of claim
 52. 59. A method of making aplant that produces a fusion protein that includes a plant viral coatprotein from a single-stranded plus-sense RNA virus and a peptidecomprising amino acids MGSDGAVQPDGGQPAV or a fragment thereof,comprising infecting a host plant with the plant virus of claim
 53. 60.A method of making the polynucleotide of claim 20, comprising ligatingan oligonucleotide encoding a peptide having the sequenceMGQPDGGQPAVRNERAT or a fragment thereof to a viral coat protein gene.61. A method of making a recombinant plant viral genome comprising (a)inserting an oligonucleotide encoding a peptide having the sequenceMGQPDGGQPAVRNERAT or a fragment thereof into the genome of asingle-stranded plus-sense RNA virus so that said oligonucleotide isfused in frame with a plant viral coat protein gene and under thecontrol of a promoter functional in plants; or (b) ligating thepolynucleotide of claim 1 to the genome of a single-stranded plus-senseRNA virus thereby making said (recombinant plant viral genome.
 62. Amethod of making the recombinant plant virus that encodes a fusionprotein that includes a plant viral coat protein from a single-strandedplus-sense RNA virus and a peptide comprising amino acidsMGQPDGGQPAVRNERAT or a fragment thereof, comprising the steps of; (a)ligating DNA encoding the fusion protein which protein is capable ofbeing expressed in a plant or a plant cell, to a DNA copy of the genomeof a single-stranded plussense RNA virus; (b) transcribing said ligatedDNA to RNA; and (c) infecting a host plant or plant cell with saidtranscribed RNA, so that said plant or plant cell makes said recombinantplant virus.
 63. A method of making the recombinant plant virus thatencodes a fusion protein that includes a plant viral coat protein from asingle-stranded plus-sense RNA virus and a peptide comprising aminoacids MGQPDGGQPAVRNERAT or a fragment thereof, comprising the steps of;(a) ligating DNA encoding the fusion protein which protein is capable ofbeing expressed in a plant or a plant cell, to a DNA copy of the genomeof a single-stranded plussense RNA virus, under the control of apromoter that is functional in plants; and (b) transforming ortransfecting a host plant or plant cell with said ligated DNA, so thatsaid DNA is expressed in said plant or plant cell, whereby said plant orplant cell makes said recombinant plant virus.
 64. A method of making aplant cell that produces a fusion protein that includes a plant viralcoat protein from a single-stranded plus-sense RNA virus and a peptidecomprising amino acids MGQPDGGQPAVRNERAT or a fragment thereof,comprising transforming or transfecting a host plant cell with the plantviral genome of claim
 61. 65. A method of making a plant cell thatproduces a fusion protein that includes a plant viral coat protein froma single-stranded plus-sense RNA virus and a peptide comprising aminoacids MGQPDGGQPAVRNERAT or a fragment thereof, comprising infecting ahost plant cell with the plant virus of claim
 62. 66. A method of makinga plant cell that produces a fusion protein that includes a plant viralcoat protein from a single-stranded plus-sense RNA virus and a peptidecomprising amino acids MGQPDGGQPAVRNERAT or a fragment thereof,comprising infecting a host plant cell with the plant virus of claim 63.67. A method of making a plant that produces a fusion protein thatincludes a plant viral coat protein from a single-stranded plus-senseRNA virus and a peptide comprising amino acids MGQPDGGQPAVRNERAT or afragment thereof, comprising transforming or transfecting a host plantwith the plant viral genome of claim
 61. 68. A method of making a plantthat produces a fusion protein that includes a plant viral coat proteinfrom a single-stranded plus-sense RNA virus and a peptide comprisingamino acids MGQPDGGQPAVRNERAT or a fragment thereof, comprisinginfecting a host plant with the plant virus of claim
 62. 69. A method ofmaking a plant that produces a fusion protein that includes a plantviral coat protein from a single-stranded plus-sense RNA virus and apeptide comprising amino acids MGQPDGGQPAVRNERAT or a fragment thereof,comprising infecting a host plant with the plant virus of claim
 63. 70.A method of isolating a virus, comprising: (a) Homogenizingvirus-containing plant tissue in Na₂S₂O₅; (b) Straining the homogenateto obtain green juice; (c) Adjusting the pH of the green juice to 5.0with acid; (d) Heating the green juice to about 47° C. for a period ofabout 5 minutes followed by cooling to about 5° C.; (e) Centrifuging thegreen juice at about 6000×g for about 3 minutes to obtain a supernatantand pellet; (f) Precipitating the supernatant in polyethylene glycol andNaCl to obtain a precipitate; (g) Resuspending the precipitate in waterat a concentration of about 1 mg per ml; (h) Extracting the precipitatein chloroform and butanol and centrifuging the extract (i) Recoveringand lyophilizing the aqueous phase of the centrifuged material; (j)Resuspending the lyophilized material at a concentration of about 5 toabout 10 mg per ml water.
 71. A method of isolating a virus, comprising:(a) Grinding virus-containing plant material in a buffer to obtain ahomogenate; (b) Straining the homogenate to obtain green juice, andadding thereto polyethyleneimine to a concentration of about 0.1% (v/v)(c) Stirring at about 4° C. for about 30 minutes followed bycentrifuging; at about 3000×g for about 5 minutes to obtain asupernatant; (d) Precipitating the supernatant in polyethylene glycoland NaCl to obtain a precipitate; (e) Resuspending the precipitate inwater at a concentration of about 1 mg per ml; (f) Extracting theresuspended material in chloroform and butanol and centrifuging theextracted material; (g) Recovering and lyophilizing the aqueous phase;and (h) Resuspending the lyophilized material at a concentration ofabout 5 to about 10 mg per ml water.